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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Screen Quest™ Membrane Potential Assay Kit *Red Fluorescence*/36004/1 plate
产品编号:36004
市  场 价:¥56560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$2828.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Screen Quest™ Membrane Potential Assay Kit *Red Fluorescence*/36004/1 plate
商品介绍
Overview
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Ex/Em(nm)635/660
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryIonChannels
MembranePotentials
RelatedBiochemicalAssays
Membranepotentialisthedifferenceinvoltagebetweentheinteriorandexteriorofacell.Themembranepotentialallowsacelltofunctionasabattery,providingpowertooperateavarietyof"moleculardevices"embeddedinthemembrane.Inelectricallyexcitablecellssuchasneurons,membranepotentialisusedfortransmittingsignalsbetweendifferentpartsofacell.Openingorclosingofionchannelsatonepointinthemembraneproducesalocalchangeinthemembranepotential,whichcauseselectriccurrenttoflowrapidlytootherpointsinthemembrane.Ionchannelshavebeenidentifiedasimportantdrugdiscoverytargets.OurScreenQuest™MembranePotentialAssayKitisahomogeneousassaywithfastreadtime.Itusesourproprietarylongwavelengthmembranepotentialindicatortodetectthemembranepotentialchangethatiscausedbytheopeningandclosingoftheionchannels.Theredfluorescenceofthemembranepotentialindicatorusedinthekithasenhancedfluorescenceuponenteringcellsandminimizestheinterferencesresultedfromthescreeningcompoundsand/orcellularautofluorescence.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareCells:

1.1   Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.

1.2   Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinequalamountofHHBSandMPdye-loADIngsolution(seeStep2.2below)at125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.

Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization.

2.PrepareMPdye-loadingsolution:

2.1   Thawonebottleof10XMPSensor(ComponentA),andonebottleofHHBS(ComponentB)atroomtemperaturebeforeuse.

Note1:1mLof10XMPSensor(ComponentA)isenoughforoneplate.Unused10XMPSensor(ComponentA)canbealiquotedandstoredat<-20oCforafewmonthsifthebottleissealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.

Note2:HHBS(ComponentB)canbestoredat4oCforconvenience.

2.2   MakeMPdye-loadingsolutionforonecellplatebyadding1mLof10XMPSensor(ComponentA)into9mLofHHBS(CompontB),andmixingthemwell.ThisMPdye-loadingsolutionisstableforatleast2hoursatroomtemperature.

 

3.RunMembranePotentialAssay:

3.1    Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofMPdye-loadingsolution(fromStep2.2)intothecellplate.

Note1:Ifyourscreeningcompoundsinterferewithgrowthmediumandserumfactors,replacethegrowthmediumwithequalvolumeofHHBSbufferbeforeaddingtheMPdye-loadingsolution.Alternatively,cellscanbegrownunderserum-freeconditions.

Note2:DoNOTwashthecellsafterdyeloading.

3.2    Incubatethedye-loadingplateina5%CO2,37oCincubatorfor30to60minutes.

Note:Insomecases,30to60minutesroomtemperatureincubationmayworkbetter.

3.3   PreparethecompoundplatesbyusingHHBS(ComponentB)oryourdesiredbuffer.

3.4   MonitorthefluorescenceintensityatEx/Em=620/650nm(bottomread).

Note:Itisimportanttorunthesignaltestbeforetheexperiment.Differentinstrumentshavetheirownintensityrange.Adjustthesignaltestintensitytothelevelof10%to15%ofthemaximuminstrumentintensitycounts.

References&Citations
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1.   VasilyevDV,ShanQJ,LeeYT,SolovevaV,NawoschikSP,KaftanEJ,DunlopJ,MayerSC,BowlbyMR.(2009)Anovelhigh-throughputscreeningassayforHCNchannelblockerusingmembranepotential-sensitivedyeandFLIPR.JBiomolScreen,14,1119.

2.   SollyK,CassadayJ,FelixJP,GarciaML,FerrerM,StruloviciB,KissL.(2008)MiniaturizationandHTSofaFRET-basedmembranepotentialassayforK(ir)channelinhibitors.AssayDrugDevTechnol,6,225.

3.   WeinglassAB,SwensenAM,LiuJ,SchmalhoferW,ThomasA,WilliamsB,RossL,HashizumeK,KohlerM,KaczorowskiGJ,GarciaML.(2008)Ahigh-capacitymembranepotentialFRET-basedassayforthesodium-coupledglucoseco-transporterSGLT1.AssayDrugDevTechnol,6,255.

4.   LiuCJ,PriestBT,BugianesiRM,DulskiPM,FelixJP,DickIE,BrochuRM,KnausHG,MiddletonRE,KaczorowskiGJ,SlaughterRS,GarciaML,KohlerMG.(2006)Ahigh-capacitymembranepotentialFRET-basedassayforNaV1.8channels.AssayDrugDevTechnol,4,37.

5.   BenjaminER,SkeltonJ,HanwayD,OlanrewajuS,PruthiF,IlyinVI,LaveryD,VictorySF,ValenzanoKJ.(2005)Validationofafluorescentimagingplatereadermembranepotentialassayforhigh-throughputscreeningofglycinetransportermodulators.JBiomolScreen,10,365.

6.   FelixJP,WilliamsBS,PriestBT,BrochuRM,DickIE,WarrenVA,YanL,SlaughterRS,KaczorowskiGJ,SmithMM,GarciaML.(2004)Functionalassayofvoltage-gatedsodiumchannelsusingmembranepotential-sensitivedyes.AssayDrugDevTechnol,2,260.

7.   JensenAA,Brauner-OsborneH.(2004)PharmacologicalcharacterizationofhumanexcitatoryaminoacidtransportersEAAT1,EAAT2andEAAT3inafluorescence-basedmembranepotentialassay.BiochemPharmacol,67,2115.

8.   JensenAA,KristiansenU.(2004)Functionalcharacterisationofthehumanalpha1glycinereceptorinafluorescence-basedmembranepotentialassay.BiochemPharmacol,67,1789.

9.   DavidLS,PlakasSM,ElSaidKR,JesterEL,DickeyRW,NicholsonRA.(2003)Arapidassayforthebrevetoxingroupofsodiumchannelactivatorsbasedonfluorescencemonitoringofsynaptoneurosomalmembranepotential.Toxicon,42,191.

10.   FitchRW,XiaoY,KellarKJ,DalyJW.(2003)Membranepotentialfluorescence:arapidandhighlysensitiveassayfornicotinicreceptorchannelfunction.ProcNatlAcadSciUSA,100,4909.


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